The overall goal is to elucidate at the molecular level the signaling pathways from the insulin receptor to the known cellular responses of insulin. Several pathways being with the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1); in one of these, IRS-1 then complexes with and activates the enzyme phosphatidylinositol 3-kinase (PI 3-kinase). One specific aim of this proposal is to determine what metabolic effects of insulin require this activation. The experimental design is to express a catalytically inactive point mutant of the 110 kD subunit of PI 3-kinase in 3T3-L1 adipocytes and in the fat tissue of transgenic mice and then to measure a variety of insulin effects, including the stimulation of glucose transport. This mutant subunit should function as a dominant negative for insulin activation of PI 3- kinase, and thereby reveal what insulin actions require this activation. The two other specific aims of this proposal are to characterize two proteins that are likely to be direct substrates for the insulin receptor and may, like IRS-1, regulate PI 3-kinase. One is a 60 kD protein that has been purified from rat adipocytes (referred to as pp60); the other is a 170 kD protein that has been slightly characterized to date in hematopoietic cell lines (referred to as 4PS). Both undergo rapid tyrosine phosphorylation in response to insulin and, in their phosphotyrosine state, complex with PI 3-kinase (at least with the 85 kD subunit thereof). Specific aims are: (i) to clone and sequence the cDNA's encoding these (ii) to describe their tissue and subcellular distributions (iii) to determine the effect of the association with PI 3-kinase upon the activity of this enzyme, both in cell lysates and with the purified proteins (iv) to discover other functions of each, through identification of other associated proteins and possibly through assay for other activities suggested by the primary sequence. The results of these studies will guide subsequent efforts to delineate the functions of these two proteins in insulin signaling in vivo. The proposed research is of direct relevance to diabetes. For both non- insulin dependent diabetes (NIDDM) and insulin-independent diabetes (IDDM) a knowledge of the signaling pathways from the insulin receptor may serve as the basis for the design of better therapeutic agents and/regimes. Moreover, in the case of NIDDM, where the basic cause(s) are not yet known, this knowledge may provide the framework for identifying the cause(s).